PROJECT SUMMARY Changes in airway epithelial cell gene transcription are important for asthma pathophysiology. Enhancers are a major class of transcription regulators but little is known about enhancer activity in airway epithelial cells or about how enhancer activity changes in asthma. We have assembled a multidisciplinary team with expertise in airway epithelial cell biology, asthma, and regulatory genomics to address this major knowledge gap. Our overall goals are to identify enhancers that are important in airway epithelial cell differentiation, to determine how enhancer activity changes in asthma, and to develop approaches for targeting the activity of these enhancers. Powerful genome-wide tools are now available for identifying epigenetic signatures associated with active enhancers (ChIP-seq, ATAC-seq), profiling transcripts in large numbers of individual cells (Drop-seq), and measuring and modulating enhancer activity (reporter assays, CRISPR). Our preliminary studies demonstrate the feasibility and utility of applying these tools to cultured primary human bronchial epithelial cells and to freshly isolated epithelial cells from individuals with asthma. We propose to use these tools to address three aims: 1) Identify candidate enhancers important for differentiation of human airway epithelial cell subsets and for responses to cytokines relevant to asthma, 2) Identify differences in airway epithelial epigenetic enhancer signatures in asthma, and 3) Determine the function of candidate enhancers in airway epithelial cells and reprogram the epithelium by targeting selected enhancers. In addition to advancing our understanding of the mechanisms of gene regulation in health and disease, this work will be invaluable for helping to explain how genetic variation in non-coding regions can contribute to asthma susceptibility and will set the stage for new highly targeted approaches for therapeutic reprogramming of the airway epithelium in asthma.